All tools predestinated for use in RNA isolation and Northern blot analysis were incubated in 50 mM NaOH solution for 20 min in order to inactivate RNAses.
Heat resistant solutions, glass and plastic materials were sterilized by autoclaving for 25 min at 121°C. Non heat resistant solutions were sterilized by filtration (filter pore size 0.2 μm).
2.2.2 Isolation of nucleic acids
2.2.2.1 Mini-preparation of plasmid DNA
Buffers required for mini-preparation of plasmid DNA:
Resuspension buffer (pH 8.0) 50 mM Tris 10 mM EDTA
0.1% RNAse A
Lysis buffer 200 mM NaOH
1% SDS
Neutralization buffer (pH 5.5) 3 M potassium acetate
Mini-cultures of E. coli bearing a plasmid of interest were received by transferring an E. coli colony from the agar plate to 5 ml LB-medium supplied with a proper antibiotic for selection. The culture was shaken at 750 – 900 rpm, 37°C for 8 – 12 h. The isolation of plasmid DNA from culture was performed according to a modification of the alkaline lysis method (Birnboim and Doly, 1979). Two ml of the mini-culture were centrifuged for 10 min at 1780 x g, 4°C. The pellet was resuspended in 100 μl RNAse A-containing resuspension buffer, incubated for 5 min with 200 μl alkaline lysis buffer and then for another 5 min with 150 μl neutralization buffer. Protein precipitate and cell debris were sedimented by
centrifugation at 21910 x g, 4°C for 30 min. The supernatant was transferred into a new 1.5 ml Eppendof cup, carefully mixed with 2 volumes of 100% ethanol (1 ml) and centrifuged at 21910 x g, 4°C for 20 min. The received DNA pellet was washed with 1 ml 70% ethanol, centrifuged at 21910 x g, 4°C, for 5 min, dried in the vacuum centrifuge at RT and dissolved in 15-20 μl autoclaved bidistilled water. The obtained plasmid DNA was used for restriction analyses, sequencing or eukaryotic cell transfection.
2.2.2.2 Preparation of E. coli stocks
E. coli stocks were made from mini-cultures (chapter 2.2.2.1) by pipetting up and down 300 μl of 80% glycerol together with 300 μl E. coli suspension. This mixture was immediately transferred to -20°C and after 24 h to -80°C.
2.2.2.3 Endotoxine-free maxi-preparation of plasmid DNA
Due to the need of a larger amount of plasmids for transfection of eukaryotic cells, plasmids were isolated using a maxi-prep kit (Endo-Free Plasmid Maxi Kit, Qiagen). For the starter culture approximately 20 μl of bacteria from the plasmid carrying E. coli stock were inoculated into 5 ml of LB medium containing the appropriate antibiotic and incubated for approximately 8 h at 37°C with shaking. 150 ml of LB medium containing the appropriate antibiotic were inoculated with 150 μl of the starter culture and grown at 37°C for 12-16 h with vigorous shaking. The over-night E. coli culture was centrifuged at 3220 x g for 20 min, 4°C (5810R, Eppendorf) and lysed according to the protocol present in the kit. Neutralization of the lysis reagent yielded a precipitate containing genomic DNA, proteins and salt complexes. The mixture was applied to the QIAfilter Maxi Cartridge. The flow-through containing the plasmid DNA was further purified through an equilibrated anion exchanging silica column, QIAGEN-tip 500. The purified DNA was eluted, mixed with 0.7 volume of isopropanol and centrifuged at 17000 x g (J2-21M/E centrifuge), at 4°C for 30 min. The received pellet was washed with 70% ethanol, centrifuged at 17000 x g at 4°C for 20 min, dried in open air and dissolved in 30 – 80 μl of endotoxin-free TE buffer (Qiagen). For DNA concentration measurements 1:1000 DNA dilutions in bidistilled autoclaved water were used. The plasmid DNA was frozen at -20°C.
2.2.2.4 Isolation of total cellular RNA
Due to different applications of RNA (RT-PCR, Northern blot) and sources (cell line or rat organ) different methods were used for isolation of total cellular RNA: the RNeasy Mini Kit (Qiagen), isolation using TRIzol™ reagent and a modification of the method described by Chomczynski and Sacchi (1987).
2.2.2.4.1 RNA isolation according to Chomczynski and Sacchi
The GTC solution was used to disrupt cells for RNA isolation from cultured mammalian cells and rat organs according to Chomczynski and Sacchi.
GTC solution: 4 M GTC
17 mM sarcosyl
25 mM tri-sodium citrate 0.1 M β-mercaptoethanol
GTC, sarcosyl (N-dodecanoyl-N-methylglycine, sodium salt) and tri-sodium citrate were dissolved in autoclaved bidistilled water at 50°C. The pH was adjusted at room temperature to 7.5 and the solution was subjected to filtration through a 0.45 μm pore size filter. The solution could be stored at 4°C for up to three months. Before usage, 1% of β-mercaptoethanol was added to the solution.
2.2.2.4.1.1. RNA isolation from cultured mammalian cells
Five x 106 to 1 x 107 cells (two 58 cm2 culture plates) per sample were used for RNA extraction. Following culture, cells were washed with PBS and the culture plates shock frozen in liquid nitrogen. One ml of GTC solution was applied per frozen cell plate. Lysed cellular material was scraped from the plates with a rubber policeman and transferred into 13 ml polypropylene centrifuge tubes. Both plates were additionally washed with 0.5 ml of GTC solution in order to reduce the material loss to a minimum. At the end, the volume of
GTC solution amounted to 3 ml per sample. 0.3 ml sodium acetate solution (pH 4.0) were added to the cell-GTC solution suspension and gently mixed by inverting the tube. The addition of 3 ml phenol (water saturated, pH 4.0) was followed by the addition of 0.6 ml chloroform isoamyl alcohol (49:1) solution to the sample. The mixture was shaken vigorously and left on ice until two phases appeared (10 – 30 min). The centrifugation of the sample at 7740 x g for 20 min at 4°C in the JA 20 rotor resulted in focussing of a protein precipitate between the two phases. The upper, RNA containing phase was transferred into a new tube, mixed with 3 ml isopropyl alcohol and kept at -20°C for at least 3 h. The centrifugation of the sample at 15,000 x g and 4°C for 30 min yielded a pellet that was dissolved in 1.5 ml GTC solution. After addition of 1.5 ml isopropyl alcohol, the sample was mixed, incubated at -20°C for at least 3 h and centrifuged at 15,000 x g, 4°C for 30 min. The obtained pellet was washed with 1 ml ethanol in order to get rid of salts, dried and dissolved in 20-40 μl of autoclaved bidistilled water containing 2.5% of 80 mM DTT (Applichem) solution and 2.5% of RNase Block (Stratagene). The sample was shock frozen in liquid nitrogen and stored at -80°C.
2.2.2.4.1.2. RNA isolation from rat organs
Isolated rat organs were cut into pieces with scissors and frozen in liquid nitrogen. Organ parts were crushed into fine powder in a pre-chilled mortar plate under constant freezing with liquid nitrogen. One fine spoon (micro spoon: 7 x 5 mm, Ochs GmbH, Bovenden-Lenglern) of the powder was mixed with 3 ml GTC solution in a sterile polypropylene centrifuge tube. The received homogeneous suspension was subjected to the following RNA extraction steps as described in chapter 2.2.2.4.1.1.
2.2.2.4.2 Total RNA isolation using TRIzol™ reagent
The TRIzol reagent is a ready-to-use reagent for the isolation of total RNA from cells and tissues. The reagent, a mono-phasic solution of phenol and guanidine isothiocyanate, is an improvement to the RNA isolation method developed by Chomczynski and Sacchi, 1987.
One ml of TRIzol reagent was used to lyse 5 x 106 cells by pipetting up and down. The
sample was incubated at room temperature (RT) to permit the complete dissociation of nucleoprotein complexes. Following addition of 0.2 ml of chloroform (per 1 ml of TRIzol reagent), the tube was capped securely and shaken vigorously by hand for 15 seconds. The sample was incubated for 2-3 min at RT and then centrifuged at 12,000 x g for 15 min at 4°C. The upper aqueous phase containing the RNA was transferred into a fresh tube and mixed with 0.5 ml of isopropyl alcohol per 1 ml of TRIzol reagent used for the initial homogenization. The sample was incubated for 10 min at RT and centrifuged at 12,000 x g for 10 min at 4°C. The obtained RNA pellet was washed by vortexing with 1 ml 70% ethanol (per 1 ml of TRIzol) and centrifuged at 7,500 x g for 5 min at 4°C. The pellet was air-dried for 5 - 10 min (retaining residual moisture), dissolved in 20 - 40 μl of autoclaved bidistilled water by passing the solution a few times through a pipette tip, and incubated at 55°C for 10 min.
2.2.2.4.3 RNeasy Mini Kit
RNA isolation using the RNeasy Mini Kit was performed according to the RNeasy Spin protocol with 4 - 5 x 106 cells (one 58 cm2 plate) per sample. All following steps were performed at room temperature (20°C between 25°C) including centrifugation steps. The medium was completely aspirated and the cells washed with PBS. Six hundred μl of lysis buffer were added to the cells and the cell lysate was collected with a rubber policeman. The lysate was pipetted into a 1.5 ml Eppendorf tube and vortexed. One volume of 70% ethanol was added to the lysate and mixed well by pipetting. The lysate was applied to the RNeasy mini column placed in a 2 ml collection tube in 700 μl portions and centrifuged at 8,000 x g for 15 min. The flow-through was discharged. The column was washed once with 700 μl RW1 buffer and then twice with 500 μl of RPE buffer. Every wash was followed by 15 seconds centrifugation at 8000 x g and the flow-through was discharged. The RNA was eluted in 30 - 50 μl of RNase-free water through centrifugation for 1 min at 8,000 x g and stored at -80°C.
Extracted RNA samples were diluted 1:1000 for the measurement of RNA concentrations.
2.2.2.5. Determination of nucleic acid concentrations
The photometric concentration measurement of both DNA and RNA was performed using the BioPhotometer (Eppendorf). The UV light absorbance was measured at 230 nm, 260 nm and 280 nm of nucleic acid samples and the ratios A260/A280 and A260/A230 were calculated.
The factor for the conversion of UV absorbance into nucleic acid concentration in μg/ml is 50.0 for DNA and 40.0 for RNA, meaning that if the light absorbance at 260 nm equals 1.0, then there are 50 μg/ml of dsDNA or 40 μg/ml of ssRNA in the measured sample. On the basis of this definition the UV light absorption of nucleic acid samples was measured and the received OD (optical density) calculated into μg/ml. The ratios A260/A280 and A260/A230
indicate the contamination degree of sample measured with proteins and salts, respectively.
The light absorbance at 230 nm of high purity samples should be approximately zero and the A260/A280 should be above 1.7, whereas the ratio A260/A230 should be beneath 2.0.